Helicobacter pylori (H. pylori) was reported to be associated with reduced effectiveness of anti-PD1 immunotherapy in patients with non-small-cell lung cancer (NSCLC). Gastric cancer is more closely related to H. pylori, so we conducted a retrospective analysis to verify whether the association of H. pylori and effectiveness is applicable to advanced gastric cancer (AGC) patients. Accumulating evidence has revealed that the gut microbiota influences the effectiveness of immune checkpoint inhibitors (ICIs) in cancer patients. As a part of the human microbiome, H. pylori and received anti-PD-1 antibodies were enrolled in the study. The differences in the disease control rate (DCR), overall survival (OS) and progression-free survival (PFS) between the H. pylori-positive group and the negative group were compared.AGC patients who had evidence of H. pylori positive, and the prevalence of H. pylori infection was 44.2%. Compared with the H. pylori-negative group, patients in the H. pylori-positive group had a higher risk of nonclinical response to anti-PD-1 antibody, with an OR of 2.91 (95% CI: 1.13–7.50). Patients in the H. pylori-negative group had a longer OS and PFS than those in the positive group, with an estimated median OS of 17.5 months vs. 6.2 months  and a median PFS of 8.4 months vs. 2.7 months . Multivariate analysis indicated that H. pylori infection was independently associated with PFS .A total of 77 patients were included in this study; 34 patients were H. pylori infection is associated with the outcome of immunotherapy for AGC patients. Multicenter, large sample and prospective clinical studies are needed to verify the association.Our study unveils for the first time that  Gastric cancer is the fifth most common cancer and third most common cause of cancer-related death worldwide after lung and colorectal cancers . AlthougHelicobacter pylori (H. pylori) is one of the most widespread bacterial pathogens worldwide, with an approximately 50% prevalence in the global population. Chronic infection with H. pylori is the main cause of gastric cancer, accounting for approximately 89% of distal gastric cancer cases worldwide [H. pylori colonizes the surface of the stomach mucosa and is not only closely associated with many disorders of the upper gastrointestinal tract but also related to some diseases localized outside the stomach [H. pylori infection [H. pylori may alter the balance of immunomodulation. Therefore, it is reasonable to speculate that H. pylori may influence the response to cancer immunotherapies. However, there are very few clinical studies in this area, and thus far, we have not seen any relevant reports on the relationship between H. pylori and immunotherapy of gastric cancer.Accumulating evidence has revealed that the gut microbiota has a considerable influence on the effectiveness of checkpoint blockade immunotherapy in human cancer patients –7, and torldwide , 14. Theorldwide . H. pylo stomach –18. In cnfection –21. ThesH. pylori-positive group and the H. pylori-negative group to evaluate the association of H. pylori infection with outcomes in AGC patients treated with anti-PD-1 antibodies.In this study, we compared the differences in disease control rate (DCR), OS and PFS between the H. pylori examination. Exclusion criteria: Subjects received only one cycle of ICIs therapy, and clinical data were not available or were lost to follow-up.This was a retrospective study conducted with a cohort of AGC patients treated with ICIs at General Hospital of Chinese PLA between May 2015 and June 2020. The inclusion criteria were as follows: 1) patients with pathologically or cytologically confirmed AGC, including cardia and noncardia gastric cancer; 2) patients treated with anti-PD-1 antibody or CTLA-4 antibody; 3) all patients with evidence of H. pylori examination, all patients should be tested for H. pylori prior to ICIs initiation. The diagnostic methods for H. pylori infection include the 13C-urea breath test (13C-UBT), H. pylori stool antigen (HpSA) test and histopathology. 13C-UBT was performed in the morning after fasting for at least 8 h. Breath samples were collected from each subject at baseline and 30 min after drinking 70 mL of water containing 75 mg of 13C-urea. An additional breath sample was collected 30 min after the ingestion of the tracer. The test was performed with a 13C-breath test instrument . The results were defined as positive when the delta over baseline (DOB) was > 4‰. Fresh stool samples were used for the HpSA test. According to the manufacturer’s instructions, a one-step chromatographic immunoassay known as the CerTest H. pylori Blister Test (CerTest Biotec S.L.) was applied for the analysis. Based on the condition of the control line and sample line, the samples were divided into three types: positive, negative and intermediate. All intermediate data were excluded from the analysis. The endoscopic diagnosis and pathological diagnosis of H. pylori were performed by experienced endoscopy doctors and pathologists, respectively.To avoid the effect of anti-PD-1 antibody or CTLA-4 antibody on The following data of subjects were collected from the medical records: age, sex, Eastern Cooperative Oncology Group Performance Status (ECOG PS), pathological type, primary tumor site, tumor differentiation, treatment line, anti-PD-1 agent, response rate and so on. All procedures performed in this study involving human participants were in accordance with the ethical standards of the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards .H. pylori-positive group and the H. pylori-negative group were compared.Tumor assessment was performed at baseline and then after every two treatment cycles, which was generally after every 6 weeks. According to the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines (version 1.1), clinical responses were categorized as complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The DCR was defined as patients with CR, PR, or SD. OS was calculated from the date of first immunotherapy administration until death due to any cause or up to the end of the follow-up. PFS HR was defined as the time from the date of first immunotherapy administration to the date of disease progression or death due to any cause before progression. OS and PFS that were not reached were considered censored data. The line of immunotherapy for AGC patients was classified as follows: ICIs was included in the first antitumor therapy after the diagnosis of advanced gastric cancer (lines of immunotherapy < 2), otherwise it’s defined as non-first-line therapy (lines of immunotherapy ≥ 2).In the absence of clinical progression of disease, if other anti-cancer agent that is part of a systemic anti-cancer therapy is discontinued due to toxicity and substituted by another anti-cancer agent of the same class, retain the same line of therapy. Irrespective of clinical progression of disease, if the dose or schedule of administration of one or more anti-cancer agent of an ongoing systemic anti-cancer therapy is modified for any reason, retain the same line of therapy.The differences in DCR, OS and PFS between the P value < 0.05 was considered statistically significant. GraphPad Prism 7.0  and SPSS 22.0  were used for statistical analysis.Categorical data are expressed as numbers and percentages, and the groups were compared using the chi-squared test. Survival curves for each group were estimated using Kaplan–Meier curves and compared by the log-rank test. A Cox proportional risk regression model was used for the multivariate analysis. The results are presented as hazard ratios (HR) with 95% confidence intervals (CIs). Two-sided H. pylori results were not reliable in four patients. Finally, a total of 77 patients were included in this study with a median age of 58.0 years (range: 24–88). Among them, 54 patients (70.1%) were male, and 23 patients (29.9%) were female. The ECOG PS was 0–1 for 62 patients (80.5%), and 66 patients (85.7%) were stage IV when they were diagnosed with gastric cancer. Regarding the histopathology type, 73 patients (94.8%) had adenocarcinoma, 1 patient (1.3%) had squamous carcinoma, and 3 patients (3.9%) had carcinoid carcinoma. In 15 patients (19.5%), the primary site was the gastroesophageal junction, and in 62 patients (80.5%), the primary site was the stomach. The anti-PD-1 antibodies used were nivolumab , pembrolizumab , and camrelizumab/toripalimab/tislelizumab . Forty-four patients (57.1%) had undergone gastrectomy before immunotherapy, and 53 patients received immunotherapy in combination with other treatments, including chemotherapy and targeted therapy. Among these 77 patients, 60 patients received the HpSA test, 11 patients received 13C UBT, and 6 patients were diagnosed by histopathology. The prevalence of H. pylori infection was 44.2% in this cohort. The baseline characteristics and response rate are summarized in Table A total of 95 patients were available in this study. Among them, 5 patients received only one cycle of ICIs therapy, 6 patients were lost to follow-up and 3 patients with incomplete outcome data.The ients 70.% were maH. pylori-negative group, 12 patients experienced PD by the end of follow-up, while among the 34 patients in the positive group, 18 patients had PD. The DCR in the negative group and positive group were 72.1% and 47.1%, respectively (P = 0.027). Compared with the H. pylori-negative group, patients in the H. pylori-positive group had a higher risk of nonclinical response to anti-PD-1 antibody, with an OR of 2.91 (95% CI: 1.13–7.50) . A similar prognostic association was observed for PFS: we observed prolonged PFS in patients in the H. pylori-negative group compared to the positive group . Although there was a correlation between H. pylori infection and OS , it was not statistically significant (P = 0.052)  died within the follow-up period. The median OS and PFS were 11.6 months (95% CI: 7.2–15.4) and 5.2 (95% CI: 3.2–6.9) months, respectively. Patients in the % died wiThe human microbiome includes the microbes that reside in the human body, such as bacteria, viruses, fungi, and protozoa, and their genomes. Currently, a growing number of studies have evidenced the relationship between the local resident microbiota and the gut microbiome in cancer and cancer treatment –24. The H. pylori infection and tumor immunotherapy has attracted the attention of researchers. Recently, Oster P et al. found that in mice engrafted with MC38 colon adenocarcinoma or B16-OVA melanoma cells, the tumor volumes of noninfected mice undergoing anti-CTLA4 and/or PD-1 or anticancer vaccine treatments were significantly smaller than those of infected mice. Two independent cohorts of patients with NSCLC on anti-PD-1 therapy verified that H. pylori seropositivity is associated with a lower effectiveness of anti-PD-1 immunotherapy in humans [H. pylori-positive patients had a higher risk of nonclinical response to anti-PD-1 antibody, and we also observed prolonged PFS and OS in patients in the H. pylori-negative group compared to the H. pylori-positive group. To our knowledge, the present study is the first to evaluate the association of H. pylori infection with outcome in AGC patients treated with an anti-PD-1 antibody. Although Cox regression revealed that H. pylori infection was not independently associated with OS, it is still helpful to predict the efficacy and prognosis of immunotherapy for AGC patients. It should be noted that the diagnostic methods for H. pylori infection in this study include 13C-UBT, HpSA test and histopathology, which all reflect the current active H. pylori infection. This is different from the study of Oster P et al., in which the H. pylori seropositive patients included both past and current infection populations. Although they found that the eradication of H. pylori infection by antibiotic administration does not increase the efficacy of vaccine-based immunotherapy, we cannot exclude the possibility that current active H. pylori infection and past infection may have a different influence on tumor immunotherapy.Similar to the intestinal flora, the relationship between n humans . Their sH. pylori infection can affect tumor immunotherapy is not clear. According to the current research, this may be attributed to the microenvironment. Like most solid tumors, the microenvironment of epithelial-derived gastric adenocarcinoma consists of a variety of stromal cell types, including fibroblasts and neuronal, endothelial and immune cells. It is reported that H. pylori infection can prevent allergic asthma in mouse models through the induction of regulatory T cells [H. pylori inhibited antitumoural CD8+ T-cell responses by altering the cross-presentation activities of dendritic cells (DCs) in humans. They also observed a decreased number of myeloid cells and a substantially decreased expression of genes induced by type I interferon, IFNγ and IL-6 in the tumors of infected patients with NSCLC undergoing anti-PD1 treatment [H. pylori and H. pylori-derived factors on immune cells influences the effect of tumor immunotherapy. However, the impact of H. pylori infection on the composition of human gastrointestinal microbiota has been verified [H. pylori could trigger large intestinal microbiota [H. pylori, in addition to affecting immune cells, may alter the gastrointestinal microbiota to influence tumor immunotherapy. Additional experiments are needed to explore the underlying mechanisms of H. pylori in decreasing the effectiveness of cancer immunotherapies.The treatment of tumors by immune checkpoint inhibitors depends on the activation of immune cells , but the T cells . Oster Preatment . It is pverified , and it crobiota . TherefoThere were some limitations in this study. First, this was a retrospective analysis of a small sample size from a single center, so external validation cohort studies with larger sample sizes are needed to confirm the robustness of our findings.H. pylori in this study reflect the current active infection. Further research is needed to determine whether this result is applicable to past infection populations. Finally, as important prognostic factors for gastric cancer immunotherapy, the data of PD-L1 combined positive score positivity, microsatellite instability-high (MSI-H) and Epstein–Barr virus-positive (EBV +) [Second, the diagnoses of  (EBV +)  were incH. pylori infection and the outcome of immunotherapy for AGC patients. In the future, H. pylori may become a powerful prognostic biomarker of personalized immunotherapy for cancer patients. However, multicenter, large sample and prospective clinical studies are needed to verify the association. The role of H. pylori in predicting prognosis in patients treated with anti-PD-1 antibody, and the underlying molecular mechanisms need further study.In summary, our study is the first to show the association between


Caenorhabditis elegans, which belongs to the superfamily of G-protein coupled receptors (GPCRs), mediates most of the physiological effects of extracellular adenosine. Adenosine has been proved to improve the survival rate of C. elegans in oxidative stress conditions. However, the potential mechanism of adenosine's protective effect against oxidative stress via AdoR-1 has not been studied. In this study, C. elegans were divided into three groups: two groups with paraquat treatment, one in the presence and one in the absence of adenosine, and an untreated control group. Results indicate that many differentially expressed genes were found to be enriched significantly in neural-related signaling pathways among transcriptome data of three groups. Further gene network analysis showed that some important genes well known to be involved in promoting the acetylcholine release pathway, such as dop-1, egl-30, and unc-13, and those involved in promoting the neuropeptide release pathway, such as kin-1, were upregulated by paraquat induction but downregulated after adenosine treatment. Meanwhile, a completely opposite trend was observed for the goa-1 gene that inhibits the acetylcholine-release and neuropeptide-release pathway. Additionally, some biochemical assays including SOD, GSSG, GSH, and AChE were measured to identify the potential protection of adenosine against oxidative stress between wild-type strain N2 and ador-1 gene knockout strain EG6890. Conclusively, our study revealed series of adenosine receptor-mediated genes in C. elegans that might act as regulators of paraquat-induced oxidative stress and may indicate adenosine's promising protective effects.AdoR-1, the single adenosine receptor homolog in  It is well documented that adenosine (ADO) has an impact on the treatment of pathological conditions, such as oxidant injury in neural cells, that functions through adenosine receptor-mediated signaling pathways . AdoRs, Caenorhabditis elegans, and many genes and signaling pathways have been shown to be conserved between nematodes and humans [C. elegans and vertebrates [C. elegans that is expressed by the ador-1 gene, is known to be an ortholog of human A2BR (adenosine A2b receptor).The first completely sequenced genome of a multicellular organism was that of d humans . In parttebrates . AdoR-1,C. elegans in oxidative stress conditions [Previous evidence has shown that Drosophila AdoR mutants have significantly increased sensitivity to the oxidative stress caused by paraquat . Our labnditions  However,nditions . Thus, iC. elegans [C. elegans groups treated with paraquat in the presence or absence of adenosine were analyzed to explore gene expression during paraquat-induced oxidative stress, and the biochemical assays from wild-type and ador-1 gene knockout strains treated with paraquat in the presence or absence of adenosine were also measured to evaluate the protective effect of AdoR-1. The results were used to uncover the molecular mechanism driving the protective effect of the nematode AdoR-1 after the addition of adenosine in these conditions.Paraquat (PQ), also known as 1,1′-dimethyl-4,4′-bipyridinium dichloride, is a common chemical substance used for generating an oxidative stress in the laboratory, particularly when used to treat the nematode  elegans , 10. TheC. elegans used in the present study were wild-type Bristol (N2), originally obtained from the Caenorhabditis Genetic Center . They were maintained on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20°C as described in a previous study [The us study .2HPO4, 6 g, KH2PO4, 3 g, NaCl, 5 g, and MgSO4·7H2O, 0.25 g) based on a previous study, where the final treatment concentrations were 150 mM and 3,000 μM, respectively [Paraquat and adenosine  were dissolved in 10 mL sterilized M9 buffer (NaμM), which were abbreviated as the PQ and PQ_AD groups, respectively. Sterilized M9 buffer solution treatment was used as a control (CK group). Three groups of nematodes were then treated with the corresponding conditions for 5 minutes prior to washing with M9 buffer solution three times to remove the bulk of residual bacteria. Then, the samples were transferred to 1.5 mL tubes, and excess supernatant was removed via centrifugation . The samples were frozen immediately in liquid nitrogen for 5 min and then stored at −80°C. Total RNA was extracted immediately using TRIzol  according to the manufacturer's protocol.A mixed-population plate with gravid adults that had laid plenty of eggs was used to directly harvest eggs. Eggs were then transferred to fresh plates using a sterile pick for incubation at 20°C. Age-synchronous populations of L4-larval nematodes were obtained at 20°C for ~48 h as described in a previous study . ApproxiC. elegans, the data were divided into two comparisons: CK versus PQ (comparison A) and PQ versus PQ_AD (comparison B). An index of the reference genome was then constructed using Hisat2 v2.0.5, and paired-end clean reads were aligned to this reference genome using Hisat2 v2.0.5 [P values to control the false discovery rate. Corrected values of FGR ≤ 0.001 and fold change ≥ 2 were set as the threshold for significantly differential expression. GO enrichment analysis of differentially expressed genes was achieved using the clusterProfiler R package with an adjusted value of P < 0.05 [RNA transcriptome sequencing analysis was performed by BGI Genomics Corporation  using Illumina HiSeq technology. Reference genome and gene model annotation files were downloaded from WormBase (WS269). To assess molecular signaling pathway changes after paraquat treatment in the presence or absence of adenosine in 2 v2.0.5 –15. Feat2 v2.0.5 . Then, t2 v2.0.5 . The BenP < 0.05 .https://www.ncbi.nlm.nih.gov/tools/primer-blast/), and primer synthesis was performed by Sangon Bioengineering Co., Ltd. . Total RNA was used to synthesize cDNA with the PrimeScript™ RT reagent Kit with gDNA Eraser . Single-stranded cDNA was amplified by comparative qRT-PCR using Takara TB GREEN Premix Ex Taq II reagent  on a Bio-Rad CFX 384 real-time PCR instrument. Eleven differential genes in our GO enrichment and six genes from the G-protein signaling pathway were analyzed using CFX maestro Version 4.1 . Gene expression levels were calculated using the 2−ΔΔCt method [act-1 as an endogenous control gene. Student's t-test was performed to statistically compare the gene expression of qRT-PCR results.Based on a comparison of key differential genes in CK vs. PQ and PQ vs. PQ_AD from our transcriptome data, qRT-PCR was used to verify the expression of differential genes from our GO enrichment analysis and the mRNA expression of G-protein signaling pathway genes. Primers for each gene and the PCR procedure parameters are listed in Tables t method . The ΔCthttp://cbl-gorilla.cs.technion.ac.il/) using default parameters.Differential gene lists in the comparison of CK vs. PQ vs. PQ_AD were examined using Gene Ontology term enrichment by GOrilla (https://string-db.org on 12/19/2021). STRING transmits experimental and predictive interaction-founded information, and the interaction generated via the online tool is defined with 3-dimensional structures, auxiliary information, and confidence scores. The confidence score in the current study was set as 0.900, utilizing the STRING platform.Interaction evidence of the AdoR-1 protein from the STRING database v11.5  was usedador-1 gene during the PQ and PQ_AD treatment on the C. elegans, EG6890 strain, ador-1(ox489), kindly supplied from Dr. Erik Jorgensen laboratory , was evaluated for its biochemical activity with wild-type N2. The EG6890 strain has a deletion from 1 kb upstream and the first three exons of the ador-1 gene and was outcrossed six times. The synchronized nematodes treated individually with same above conditions  were harvested and washed free from bacteria using M9 buffer as described above.In order to prove the effect of An extract of nematodes was prepared by ground in liquid nitrogen with sterile mortar and pestle, repeated freezing and thawing twice. Homogenates were centrifuged at 12,000 × g, 4°C for 10 min, and the supernatant fractions were assayed for protein concentration . Superoxide dismutase (SOD), reduced glutathione (GSH), oxidized glutathione (GSSG), and acetylcholinesterase (AChE) activities were detected using commercial kits  according to the manufacturer's instruction. All experiments were performed in triplicate.http://www.ncbi.nlm.nih.gov/bioproject/809866.A summary of data quality assessment results from our RNA-seq data is shown in Supplementary Table Using DESeq-based statistical inference, a total of 2,537 genes were found to be differentially expressed in pair-wise comparisons of two groups, including 1878 upregulated and 391 downregulated genes in a comparison of the CK group with the PQ group, and 213 upregulated and 975 downregulated genes in a comparison of the PQ group with the PQ_AD group, respectively . Of thesdop gene family, the neuropeptide-related gene family , the neuropeptide receptor gene family (such as npr), and the acetylcholine release-related unc gene family.We next performed GO (gene ontology) analysis on the differentially expressed genes identified by RNA sequencing in the CK vs. PQ and PQ vs. PQ_AD groups Figures . We founDOP (DOPamine receptor), FLP (FMRF-like peptide), NLP (neuropeptide-like protein), and NHR (nuclear hormone receptor). From the overall trend, the qPCR verification results of the relative expression of differential genes were consistent with our RNA-seq results  and the gene related to the promotion of the neuropeptide release pathway (kin-1) showed the same trend that their expression was upregulated during PQ treatment but downregulated during PQ_AD treatment (treatments with PQ in present of adenosine) . Interestingly, although the expression of the ador-1 gene showed the same trend as dop-1, egl-30, unc-13, and kin-1, which is demonstrated in P > 0.05). This result suggested that the adenosine receptor was consistently and stably expressed regardless of whether the worms were PQ treated or not. In order to assess the dominance of the G-protein signaling pathway on adenosine protection effects, the gene (goa-1) that inhibited the above-mentioned pathways, including the acetylcholine release and neuropeptide release pathways, was also verified by qPCR. A completely opposite trend was observed for goa-1 gene expression as was observed right after PQ treatment, and this gene decreased by 65.3% while increasing to 74.5 ± 8% after adenosine addition , and included 10 proteins. Detailed description of protein data is provided in Supplementary Table dop-1, dop-3, and dop-5 genes were higher than other genes, which were consistent with the interaction prediction of literature database.Using AdoR-1 as a seed, a total of 10 protein interaction maps were constructed using the STRING version 11.5 database. The interactions based only on text mining were also verified using the PubMed literature database. The results of this analysis of protein-protein interaction networks were highly connected network of molecules .Based on our PPI network and qPCR data on members of the G-protein signaling pathway including ador-1 gene was knockout , neuropeptide-related genes , neuropeptide receptor genes (NPR), and genes related to acetylcholine release (UNC), seemed to be relevant among those identified, based on their gene functions.Keeping an FDR ≤ 0.001 and an FC value ≥2 as the thresholds using our transcriptome data, CK vs. PQ revealed 2,269 differential genes, with 1,878 being upregulated and 391 being downregulated. In contrast to this, PQ vs. PQ_AD comparison yielded 1,188 differential genes, 213 of which were upregulated, and 975 were downregulated. As an important molecule, it seems that adenosine has a direct impact on kinds of genes that regulate their expression during paraquat exposure. Although numbers of differential expression genes decreased in presence of adenosine in paraquat-exposed e stress . Furthere stress . Based oe stress , includiDOP, FLP, and NLP gene families were significantly differentially expressed  and D2-like (DOP-3), as is demonstrated in dop-1, dop-3, and dop-5 genes have a higher score than other genes. DOP-1 and DOP-3 antagonize movement by acting on the same motor neurons, which coexpress receptors and do not have postsynaptic connections with dopaminergic neurons [αq signaling pathway encoded by EGL30 and EAT-16 to regulate behavioral choice in cholinergic neurons, while DOP-3 may act through the Gαo signaling pathway encoded by goa-1 and egl-30 to regulate behavioral choice in GABAergic neurons, RIC neurons, and SIA neurons [dop-1 and dop-3 were both upregulated under paraquat-induced stress. In other words, dop-1 was upregulated, 4.12 fold relative to control, and dop-3 was upregulated, 1.15 fold relative to control. Clearly, the pathway of promoting the release of acetylcholine represented by dop-1 was dominant under paraquat-induced stress. In addition, GOA-1, which is the downstream effector molecule of DOP-3, was expressed to only 0.35 fold of the CK group under paraquat-induced stress. Therefore, this indirectly indicated that the acetylcholine-promoting pathway represented by dop-1 was dominant to dop-3, which represents the inhibition of the release of acetylcholine under paraquat-induced stress. Subsequently, the relative expression of dop-1 and dop-3 in the PQ_AD group was significantly reversed compared to the PQ group (P < 0.01). It seems that the activation of AdoR-1 reduced the content of DAG by regulating dopamine via DOP-1 and the molecular pair downstream of DOP-3, thereby reducing the release of the neurotransmitter acetylcholine. It is well known that the excessive release of acetylcholine is inherently toxic and will cause muscle cells to continue to be rigid [C. elegans treated with paraquat have been observed to be rigid and transparent, but this phenotype yielded a longer protective effect with preconditioning with adenosine, as shown in our previous study [It is well known that dopamine coregulates neuronal activity in  neurons . Moreove neurons . Our quabe rigid . This mius study .αq, Gα12, Gαo, and Gαs) that act within motor neurons to control the activity of NMJs. The Gαq, Gα12, and Gαo pathways converge to control the production and destruction of the lipid-bound second messenger diacylglycerol (DAG) at sites of neurotransmitter release. DAG acts via at least two effectors, mUNC13 and PKC, to control the release of both neurotransmitters and neuropeptides from motorneurons. The Gαs pathway converges with the three other heterotrimeric G-protein pathways downstream of DAG to regulate neuropeptide release. Released neurotransmitters and neuropeptides then act to control the contraction of the body-wall muscles to control C. elegans locomotion [Many of the proteins that regulate activity at the neuromuscular junction (NMJ) participate in a network of heterotrimeric G-protein signaling pathways controlling the release of synaptic vesicles and/or dense-core vesicles (DCVs) . There acomotion .kin-1 gene can activate cAMP-dependent protein kinase activity and is an ortholog of human PRKACA . The increase of its expression level can promote an increase in neuropeptide levels. Similarly, the expression of kin-1 in the PQ group was shown to be upregulated 3.23 fold compared to the CK group, while the average expression in the PQ_AD group was only 1.46 fold compared to the CK group. Adenosine treatment reduced this expression of kin-1 to close to 50%. As is well known, the release of neuropeptides as a regulator can change the frequency of muscle oscillations in nematodes, thereby controlling the speed and direction of their movement. In contrast, this behavior is related to nociception and feeding. Therefore, another mode of AdoR-1 action may be through regulating the expression level of kin-1 (PKAc) and regulating the release of neuropeptides, thereby alleviating the continuous contraction of muscle cells, reducing nociceptive perception and exercise frequency, thus achieving a protective effect, as is shown in Another mode of AdoR-1 action may be through regulating the expression level of KIN-1 (PKAc) and regulating the release of neuropeptides. Corresponding to the human homologous cAMP-PKAc pathway, the C. elegans may be through the continuous activation of the adenosine receptor homolog AdoR-1, the reduced release of acetylcholine, and the expression of key genes in the neuropeptide signaling pathway, acting to reduce acetylcholine. The release of neurotransmitters and neuropeptides relieves the toxicity of excess acetylcholine, and in body wall muscles, acetylcholine causes muscle contractions, slowing exercise frequency and nociceptive perception.Based on the above, the protective mechanism of adenosine against oxidative stress in C. elegans, and studies on the four adenosine receptors  in humans will benefit this study's findings on the evolution of paraquat-induced stress modulation.Here, we provide a model  of the pador-1 gene knockout strain EG6890. It is known that glutathione is an important antioxidant substance in cells; oxidized glutathione (GSSG) and reduced glutathione (GSH) are converted into each other under the catalysis of enzymes [Furthermore, in order to identify the potential factors involved in the protective mechanism of adenosine against oxidative stress, some biochemical assays were carried out between wild-type strain N2 and  enzymes . The conador-1 gene-knockout strain EG6890, the ratio of GSH/GSSG in the body is also low, indicating that GSH in the body is involved in the reduction reaction of ROS. However, combined with the results of The Reduced glutathione (GSH) is considered to be one of the most important scavengers of ROS, and its ratio with oxidized glutathione (GSSG) may be used as a marker of oxidative stress. And the reduced release of acetylcholine appears during the adenosine added after paraquat treated in wild-type nematodes. Therefore, the SOD, GSH: GSSG ratio and AChE activity illustrated in DOP, FLP, NLP, and NHR were differentially expressed when C. elegans worms were exposed to paraquat in the presence or absence of adenosine, and this study was the first to show the interaction between adenosine and neuronal G-protein signaling pathways in C. elegans. Furthermore, our research revealed that the protective effect of adenosine in terms of the oxidative stress induced by paraquat was mainly regulated by AdoR-1, and that several novel genes such as dop-1, egl-30, unc-13, kin-1, and goa-1, which were involved in key G-protein signaling pathways of the C. elegans adenosine receptor, deserve additional research. Additionally, some biochemical factors including SOD, GSSG, GSH, and AChE were identified for potential relationship with protective effect of AdoR-1 between wild-type strain N2 and ador-1 gene-knockout strain EG6890. Our study provides new insights into the biological basis of the action of the adenosine receptor via RNA-seq and contributes to a better understanding of the mechanism of the adenosine receptor.Taken together, our findings showed that genes from four classes involved in


Directional deep brain stimulation (dDBS) is preferred by patients with advanced Parkinson’s disease (PD) and by programming neurologists. However, real-life data of dDBS use is still scarce. We reviewed the clinical data of 53 PD patients with dDBS to 18 months of follow-up. Directional stimulation was favored in 70.5% of dDBS leads, and single segment activation (SSA) was used in 60% of dDBS leads. Current with SSA was significantly lower than with other stimulation types. During the 6-month follow-up, a 44% improvement in the Unified Parkinson’s Disease Rating Scale (UPDRS-III) points and a 43% decline in the levodopa equivalent daily dosage (LEDD) was observed. After 18 months of follow-up, a 35% LEDD decrease was still noted. The Hoehn and Yahr (H&Y) stages and scores on item no 30 “postural stability” in UPDRS-III remained lower throughout the follow-up compared to baseline. Additionally, dDBS relieved non-motor symptoms during the 6 months of follow-up. Patients with bilateral SSA had similar clinical outcomes to those with other stimulation types. Directional stimulation appears to effectively reduce both motor and non-motor symptoms in advanced PD with minimal adverse effects in real-life clinical care. Early stage Parkinson’s disease (PD) is usually treated with oral dopaminergic medication. As the disease progresses, motor and non-motor symptoms and symptom fluctuations become more prominent despite optimal medication. This stage of the disease is considered the advanced stage, and device-aided therapies, such as intrajejunal levodopa infusion, subcutaneous apomorphine infusion, and deep brain stimulation (DBS), should be considered to alleviate symptoms and enhance the quality of life in patients with Parkinson’s disease . DBS is DBS has become one of the main treatment options for patients with advanced PD ,3. For tSince its introduction in the mid-2010s, directional deep brain stimulation (dDBS) has been preferred by PD patients and programming physicians, even though directional programming has been reported to be more time consuming ,9,10. CoHowever, long-term real-life data on the use of directional stimulation is still scarce ,18,19. TThe data was collected retrospectively from the Helsinki University Hospital medical records and Abbott Clinician Programmer  used by the programming neurologists in the clinic. The following demographic data was obtained: patients’ age, duration of PD and advanced PD, and other medical comorbidities, including the use of anticoagulants/antiplatelet drugs and antidepressants. For this study, the Hoehn and Yahr (H&Y) stage, the use of anti-Parkinsonian medication  was colThe DBS settings , the activation of directional stimulation, and the use of individual programs were examined. Single segment activation (SSA) was compared with two segment activation (TSA) and/or omnipolar stimulation. The consumption of the IPG battery was also analyzed with different stimulation types.The patient selection for DBS treatment was assessed according to accepted guidelines . DorsolaAfter the 6-month inpatient programming visits, the patients were transferred to our outpatient clinic, with follow-up visits at 12 and 18 months. During these visits, the effect of DBS was assessed with the medication ON and stimulation ON state.t-test, Wilcoxon signed rank test, and repeated measurements ANOVA and Friedman test for repeated measurements. Bonferroni correction was used to account for multiple comparisons. p-values < 0.05 were considered significant.Because of the retrospective registry assessment, this study was approved by the director responsible for the academic research at the Helsinki University Hospital without an additional approval from ethics committee, according to the Finnish laws. The statistical analysis was performed using SPSS Statistics 27 . The data is presented as the median  and mean  when applicable. The distributions of the variables were evaluated with the Shapiro–Wilk test. Data analysis was conducted using the paired In total, 63 patients were implanted with a subthalamic dDBS system, and 62/63 operations were bilateral implantations. In total, 45 DBS operations (72%) were performed under general anesthesia and 18 (28%) under local anesthesia. The use of DBS stimulation in 125 dDBS leads was examined up to 6 months. Due to the COVID-19 pandemic and patient dropout, data of 105 dDBS leads was available for the 18-month follow-up. p < 0.001 in both). Patients also reported less non-motor symptoms as measured with NMS-Quest, as shown in p = 0.652). When each domain was separately examined, improvement was noted in the scores of the following domains: communication, bodily discomfort, and stigma (p = 0.007–0.016).Patients’ demographics are presented in p < 0.001 when 12- and 18-month scores were compared to baseline). Item no 30 “postural stability” in UPDRS-III dropped from a median of 1.0 (IQR 1.0) at baseline to a median of 0.0 (IQR 1.0) at the 6-month visit and remained stable until the end of the follow-up .After the initial 6-month programming, the H&Y stages were at a median of 2.0 (IQR 0.5) throughout the follow-up, and thus were lower than at baseline (p < 0.001), as shown in p = 0.013).After the DBS implantation, LEDD decreased and remained lower than at baseline throughout the follow-up  or a switch from directionality to omnipolarity (8.0%) at the same contact level . Other aIn the majority of leads , the stimulation type remained the same from the 6-month up to end of the 18-month follow-up. In 18/105 (17%) dDBS leads, the stimulation type was changed from directional mode to omnidirectional mode (in 7.6% of dDBS leads), SSA was changed to TSA (2.8%), or SSA changed to another SSA within the same electrode level . The modAt the end of the 18-month follow-up, in dDBS leads with tested directionality, only 17/105 (16.2%) dDBS leads had omnipolar stimulation. This accounts for five patients with bilateral omnipolar stimulation (10/17 dDBS leads), and another seven patients had omnipolarity that was unilaterally active. After the follow-up, 70.5% of all dDBS had the directional mode active and only 29.5% had the omnidirectional mode. Mostly, directional stimulation was single segment activation, as shown in p = 0.127–0.201). Additionally, the baseline UPDRS-III scores, LEDD, and H&Y stage were similar and there were no differences in the gender or advanced disease duration (p = 0.288–0.927). The LEDD or UPDRS-III reductions were similar between those that had been operated on under general anesthesia and those with local anesthesia (p = 0.433–0.822).The outcomes of patients with bilateral directional stimulation were compared to those patients with bilateral omnipolar stimulation and/or with unilateral directional stimulation and omnipolar stimulation. The changes in the UPDRS-III points and LEDD and the non-motor questionnaire or PDQ-39 scores did not differ between these groups (p < 0.001) and a median of 0.2 mA (IQR 0.6 mA) until the 18-month visit (p = 0.001 vs. 6-month visit) , with a range of 40–110 µs. The median frequency remained at 130 Hz and IQR was increased to 30.0, creating a statistical difference between baseline and the 6-, 12-, and 18-month visits (p = 0.001–0.002). The frequency ranged from 50 to 200 Hz individually. After the 6-month visit, the frequency at the 12-month or 18-month visit was similar to the frequency at the 6-month visit (p = 0.881–0.970). The median impedances rose significantly during the follow-up . When SSA was compared to TSA and/or omnipolar stimulation at the 6-, 12-, and 18-month visits, the current in SSA was significantly lower (p = 0.001–0.05) and the impedance was higher (p = 0.001–0.015) at all time points, but the frequencies or pulse widths did not differ (p = 0.144–0.720). In total, 98% of the patients had solely used one program, mostly directional, at home when examined at the 18-month visit. They presented various reasons for choosing directionality. When analyzed in this study, the patients’ UPDRS-III scores were lower with directional stimulation as compared to omnipolar stimulation .Among all stimulation types included, current increased to a median of 0.9 mA (IQR 1.0 mA) during the first 6 months (h visit) . The pulp < 0.001 at 6 and 12 months vs. at implantation). It reduced to 2.84 volts (IQR 0.03) at the 18-month visit (p < 0.001 at 18 months vs. at implantation). At the 6-, 12-, and 18-month visits, there were no statistical differences in the battery consumption between those patients who had bilateral directional stimulation and those who had either unilateral directional stimulation and/or bilateral omnipolar stimulation (p = 0.553–0.926). There was no IPG change due to battery exhaustion during the 18-month follow up.The IPG battery voltage decreased from a median of 3.0 V (IQR 0.0 V) to a median of 2.95 V (IQR 0.02 V) within the first 12 months (p = 0.819).Only one dDBS lead was reimplanted within the 12 postoperative months because of an initial suboptimal placement. Because of another patient’s complaints of discomfort, an IPG was replaced subcutaneously. After a dDBS lead implantation, the manufacturer reported a lead defect in a dDBS lead that prevented further MRI imaging but did not require a lead replacement nor did it prevent the programming. Two patients (3.2%) had deep vein thrombosis within the first postoperative month that was treated with a brief anticoagulation treatment . Four patients (6.3%) used a course of antibiotics for superficial wound infection. No severe dDBS system-related infections or operation-related hemorrhages were reported. One patient was admitted to the inpatient ward for an additional programming visit after the initial six-month inpatient programming schedule. The median score given for dysarthria in UPDRS-III was 0 points at baseline and 1 point at the 6-month visit, without a clear difference  and the impedance was higher (p = 0.001–0.015). The frequencies or pulse widths did not differ between the stimulation types. The use of SSA in this study is analogous to previous reports [p = 0.012). This suggests that in our patient cohort, patients probably had better symptom control with directionality. In the PROGRESS study, clinicians favored directionality for symptom relief in most patients (96/113) and to avoid side effects in 11/113 [n = 33), similar results were reported [Mostly directional (SSA) stimulation was used throughout the follow-up in this study. After 18 months of follow-up, 29/53 patients used bilateral directional stimulation with SSA and only 14 patients had unilaterally SSA and another dDBS lead programmed either in omnipolar mode or directional stimulation with two segments active (TSA). Only nine patients had bilateral omnipolar stimulation. The current used in SSA was lower than in other stimulation types ( reports ,24. Dire reports ,22,24. Ireported .The clinical outcomes in our study resemble prior results on alleviating Parkinsonian motor symptoms after dDBS implantation and are similar to those reported on conventional DBS treatment ,21,23,26In a study comparing PD patients with directional stimulation and omnidirectional stimulation, patients with directional stimulation had a 13% greater decline in daily anti-Parkinsonian medication than those with omnidirectional stimulation . In our Early reports on directional stimulation raised the concern of faster battery depletion due to higher impedances of directional stimulation ,33. RepodDBS has not presented greater risk in DBS lead implantation . In a twA limitation of this study is its retrospective nature and, therefore, there is a lack of comprehensive data during the follow-up. This study is single centered with its restrictions. Yet, we feel that this study gives an overview on the use of directional stimulation in a real-life setting.Directional stimulation is widely used in real-life clinical care, and it is preferred by patients when they are given the opportunity to choose. dDBS appears to be reliable and feasible in the treatment of motor and non-motor symptoms with minimal side effects in advanced PD.


Ab enzymes are intrinsic to Acinetobacter baumannii and can confer resistance to carbapenem antibiotics. Here we determined the structure of the most prevalent OXAAb enzyme, OXA-66. The structure of OXA-66 was solved at a resolution of 2.1 Å and found to be very similar to the structure of OXA-51, the only other OXAAb enzyme that has had its structure solved. Our data contained one molecule per asymmetric unit, and analysis of positions responsible for dimer formation in other OXA enzymes suggest that OXA-66 likely exists as a monomer.The OXA β-lactamases are responsible for hydrolysing β-lactam antibiotics and contribute to the multidrug-resistant phenotype of several major human pathogens. The OXA The crystal structure of OXA-66 has been deposited in the Protein Data Bank (PDB) with the PDB ID: 6T1H. The coordinate files, diffraction data and validation report can be downloaded from the PDB, DOI: 10.2210/pdb6t1h/pdb.Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii [Ab enzymes that are intrinsic to A. baumannii. It has recently been shown that these intrinsic enzymes are capable of conferring resistance to the carbapenem antibiotics [Ab enzyme OXA-51 has been solved [Ab enzyme is OXA-66 due to its association with the predominant A. baumannii Global Clone 2. We therefore sought to determine the structure of OXA-66 and compare it to OXA-51.The OXA-type β-lactamases are enzymes that hydrolyse the β-lactam antibiotics. They are commonly found in Gram-negative bacteria that cause serious infections in humans, including mannii . While sibiotics , and it n solved . HoweverA. baumannii being classed and found to be a monomer, although there is no evidence of the oligomerisation state of the full length enzyme [Although the OXA-type enzymes have been reported to have a high degree of structural similarity despite their divergence in sequence, there have been contradictory reports suggesting that the oligomerisation of these enzymes may not be universal. For example, some enzymes have been reported to be monomers such as OXA-1 where an elongated Ω-loop is postulated to inhibit the process of dimerization , or OXA-h enzyme .Ab enzyme, OXA-66, and compare it to the previously solved structures of OXA-51. Additionally we propose, based on similarity and structural models, that this sub-group of enzymes is of a monomeric nature.Here we present a new structure of the most prevalent OXAE. coli DH5α. Transformants were selected with ampicillin (100 mg l−1) and confirmed by PCR using pGEX Sequencing Primers . The recombinant plasmid was transformed into E. coli BL21 (DE3)  following manufacturer guidelines.For crystal structure determination, OXA-66 was amplified by PCR without its signal peptide, using the OXA-66-BamHI forward and reverse primers . The sigBL21 (DE3) pGEX-6P-1 OXA-66 was grown at 37 °C to an OD600 of 0.8–1.0 in LB broth before inducing with a final IPTG concentration of 0.1 mM for 6 h. A total of 11.5 g of cell pellet was yielded for purification. OXA-66 was purified according to the column chromatography method outlined by the GST-Bind Kit , up to the first flow through fraction collection. Subsequently, the manufacturer protocol for PreScission Protease cleavage of GST-tagged protein bound to the column  was followed to remove the GST-tag and elute OXA-66. Homogeneity of the purified protein was confirmed by running the eluted sample on a 10 % Bis-Tris SDS-PAGE gel . The purified protein appeared as a single band (data not shown).Octahedron crystals were grown under conditions described in The X-ray diffraction dataset was collected a wavelength of 1.5418 Å using a copper rotating anode X-ray diffraction system equipped with confocal mirror monochromator, a kappa geometry goniometer, and Platinum 135 CCD detector  at a temperature of 100K . The exposure time was set to 60 s for a single phi-oscillation image of 1 degree, and the total of 1398 oscillation images were collected in eight different kappa geometry orientations. The dataset was indexed, scaled and merged using PROTEUM2 data processing software . The resThe OXA-66 crystal structure was solved by the Molecular Replacement (MR) method. The crystal structure of OXA-51 beta-lactamase (PDB-ID: 4ZDX) was used as the MR search probe. The sequence identity between the search probe and OXA-66 is 97 %. All MR calculations were performed in PHASER, part of the PHENIX crystallographic software suite . The obtAb group, OXA-66, at a maximum resolution of 2.1 Å, solved by molecular replacement using the OXA-51 structure (PDB ID: 4ZDX) as the molecular replacement model with an amino acid sequence identity of 97 %. As expected, there is as high degree of structural similarity between these enzymes, with an R.M.S.D value of 0.48 Å on 239 c-alpha atoms with the 4ZDX apo structure of OXA-51. OXA-66 differs from OXA-51 by six amino acids – T5A, E36V, V48A, Q107K, P194Q, D225N [We present here the crystal structure of the most prevalent member of the OXAQ, D225N , but thiSimilar to the active site in the Apo OXA-51 structures (5ZKH and 4ZDX), we observe that the tryptophan at position 222 lies in a conformation disfavouring the substrate from binding in the active site  . HoweverAb enzyme has currently been classified as either a dimer or monomer. We therefore compared our OXA-66 structure with confirmed monomeric and dimeric enzymes to determine the possible oligomeric state. Three schemes of salt bridges have been identified as being important for dimerization previously with several key residues identified [One of the interesting features of the OXA enzymes is the varying reports of these highly similar enzymes being either monomeric or dimeric, although at present no OXAentified . These tentified . CompariAb enzymes. Further work using appropriate analytical methods such as size exclusion chromatography with multiple angle light scattering (SEC-MALS), or dynamic light scattering (DLS), is needed to determine the true oligomeric state of this group of enzymes.OXA-10 differs from OXA-13 and OXA-48 in that the OXA-10 salt bridges require the octahedral coordination of a metal ion to enable dimerization. Comparing the sequence and structure of OXA-66 to that of OXA-10, the hydrophobic interactions that help to promote dimerization are not present in OXA-66 . AdditioAb enzyme OXA-66 has structural similarity to OXA-51. On the bases of sequence and structural analyses, we also suggest that resides at the dimeric interface suggest the OXAAb enzymes may exist as monomers.Overall, we demonstrate that the most prevalent OXAClick here for additional data file.


Alopecurus myosuroides Huds.) is a highly competitive weed in winter wheat fields of China. Due to repeated use of acetolactate synthase (ALS) inhibitors, many A. myosuroides populations have evolved resistance to pyroxsulam in some wheat fields. Research was conducted to determine the molecular basis of herbicide resistance in the AH93 A. myosuroides population. Whole-plant dose–response assay confirmed that the AH93 population was resistant to pyroxsulam with a resistance index of 4.2. Cross- and multiple-resistance assays indicated that the AH93 population was cross-resistant to mesosulfuron-methyl and multiple-resistant to pinoxaden. Sequencing of the ALS and ACCase gene revealed that there was no target-site mutation in ALS, but Trp-2027-Cys and Cys-2088-Arg amino acid mutations in ACCase in the AH93 population. A malathion pretreatment study indicated that the AH93 population might have cytochrome P450–mediated herbicide metabolic resistance. This is the first report of pyroxsulam resistance in a multiple-resistant A. myosuroides population in China, and the Cys-2088-Arg mutation is the first reported case of an ACCase mutant conferring herbicide resistance in A. myosuroides.Black grass ( Alopecurus myosuroides Huds.) in wheat fields. However, repeated and intensive application of ALS-inhibiting herbicides poses great pressure for herbicide resistance selection in weeds. Herbicide resistance has made weed management more difficult and expensive. Therefore, clarifying the mechanism of herbicide resistance is helpful to maintain herbicide efficacy by formulating integrated resistance management strategies [The increasing number of herbicide resistance cases in weeds is a major concern for sustainable agriculture development. To control these weeds, various herbicides with different sites of action have been applied to wheat fields. Over the last decade, the acetolactate synthase (ALS)-inhibiting herbicide pyroxsulam has been extensively used to manage grass weeds such as black grass (rategies .The mechanism of herbicide resistance includes target-site-based resistance (TSR) and nontarget-site-based resistance (NTSR) mechanisms. TSR is the result of target-site mutation or overexpression of herbicide target genes . ALS-inhThe NTSR mechanism includes reducing herbicide absorption and translocation in the plant or enhancing the degradation of herbicides through plant metabolism ,8. Many A. myosuroides population could not be managed by the field-recommended rates of pyroxsulam application in the winter wheat fields in Anhui Province, China. The current study aimed to determine the pyroxsulam resistance level and investigate the resistance basis to pyroxsulam in the A. myosuroides population.In recent years, black grass has been frequently found in the Huang huai winter wheat growing areas, including the Chinese provinces of Hebei, Shandong, Anhui, and Henan, where it causes large yield reductions . Recentl−1, which was 4 times the recommended field rate. The GR50-based resistance level of the AH93 population was 4.2-fold higher than that of the TJ43 population  in eight individuals of the AH93 population. In addition, a TGC to CGC substitution at amino acid position 2088 (Cys-2088-Arg) was found in two individuals of the AH93 population  and the Trp-2027-Cys and Arg-2088-Cys amino acid mutations were identified. The Trp-2027-Cys mutation has been reported in many grass weed species, such as Digitaria insularis [Rottboellia cochinchinensis [A. myosuroides with Trp-2027-Cys mutation from France could withstand pinoxaden application in the field rate (60 g ha−1) and showed cross-resistance to fenoxaprop and clodinafop [Avena sterilis. It is important to point out that this is the first report worldwide of the ACCase mutant Cys-2088-Arg that confers target-site-resistance in black grass. In this study, both Trp-2027-Cys and Cys-2088-Arg mutation have double peaks and are heterozygous mutations. Kaundun reported that herbicide-resistant levels depend on specific amino acid changes, the number of resistant alleles, and recommended field rates of herbicides [Different ountries . To datehibitors . In thisnsularis  and Rotthinensis . A. myosodinafop . In addield rate 0 g ha−1 rbicides . FurtherIn recent years, resistant black grass populations have been established in the Huang huai winter wheat growing areas, including the Chinese provinces of Hebei, Shandong, Anhui, and Henan . What isA. myosuroides population  were collected in 2019 from a winter wheat field in Anhui Province, China. The wheat field had been treated with pyroxsulam during the year of seed collection. A known susceptible population  that had no history of herbicide application was used as a control. After air-drying, the seed samples were stored in glass bottles at room temperature.Seeds from more than 100 individual plants of the putative pyroxsulam-resistant Seeds were sown in 11 cm diameter plastic pots and grown in a greenhouse at 20 ± 3 °C for 12 h day and 15 ± 3 °C for a 12 h night with regular application of water and fertilizer. The seedlings were thinned to 8 plants per pot before herbicide treatment.−1. Pyroxsulam  was applied at 0, 0.125×, 0.25×, 0.5×, 1×, 2×, and 4× for the susceptible TJ43 and resistant AH93 populations with × = 12.6 g ai ha−1. Three weeks after treatment, the aboveground plants were cut, and the biomass was measured. The experiment was repeated two times with three replications for each treatment.When seedlings reached the 3–4-leaf stage, pyroxsulam was sprayed onto them using a laboratory sprayer equipped with a flat fan nozzle delivering 450 L ha−1 1 h prior to pyroxsulam treatment. Pyroxsulam treatments followed the same methods and rates as those used in whole-plant dose–response assays. Untreated plants and plants treated with only malathion were used as controls. At 21 days after treatment (DAT), the aboveground biomass of the plants was determined. The experiment was repeated twice with three replications for each treatment.Seedlings of the TJ43 and AH93 populations at the 3–4-leaf stage were treated with malathion or malathion plus pyroxsulam. Malathion was applied at 1000 g ai ha−1), the ACCase inhibitor pinoxaden (× = 60 g ai ha−1), or the HPPD inhibitor cypyrafluone (× = 180 g ai ha−1) at 1×, 2×, and 4× the recommended field dose (× corresponds to the recommended rate). Plant survival was recorded when plants had new growth or active tiller formation 21 days after treatment. Each herbicide treatment had four replicate pots (eight plants per pot), and the experiment was conducted twice.To estimate cross- and multiple-resistance patterns to other herbicides, seedlings of the TJ43 and AH93 populations at the 3–4-leaf stage were treated with the ALS inhibitor mesosulfuron-methyl . The ALS gene and ACCase gene were amplified according to the methods of Huang et al. [Leaves from ten individuals of the TJ43 and AH93 g et al.  and Ge eg et al. .ALS gene and ACCase gene were confirmed on a 1% agarose gel, and then each PCR product was sequenced by the Beijing Genomics Institute . Sequence data were aligned and compared using Vector NTI 12.5 .The PCR products from the t-test. Analysis of the dose–response data from the whole-plant assays were performed using SigmaPlot software . The data were fitted to a nonlinear log-logistic model:50 (the herbicide dose required for 50% growth reduction). The level of resistance was determined by the resistance ratio, which was calculated as the GR50 of the resistant population divided by that of the susceptible population.The data were expressed as a percentage of fresh weight compared to untreated control plants. Data were subjected to ANOVA and were pooled when no significant difference between the two experiments. Means of treatments were averaged and compared using Student’s For cross- and multiple-resistance, the surviving rate was recorded as a percentage of survival plants compared to untreated control plants. And each herbicide treatment contained 32 replicate individuals.


However, these effects were not observed in rats treated with 85 mM of the stereoisomer D-Gln. Our results showed that L-glutamine could specifically activate MMCs to degranulate and release MMC mediators to the lymph during fat absorption. This observation is potentially important clinically since L-glutamine is often used to promote gut health and repair leaky gut.Glutamine (Gln) is required for intestinal mucosal homeostasis, and it can promote triglyceride absorption. The intestinal mucosal mast cells (MMCs) are activated during fat absorption. This study investigated the potential role of Gln on fat absorption-induced activation of MMCs in rats. Lymph fistula rats (n = 24) were studied after an overnight recovery with the infusion of saline only, saline plus 85 mM L-glutamine (L-Gln) or 85 mM D-glutamine (D-Gln), respectively. On the test day, rats (n = 8/group) were given an intraduodenal bolus of 20% Intralipid contained either saline only (vehicle group), 85 mM L-Gln (L-Gln group), or 85 mM D-Gln (D-Gln group). Lymph was collected hourly for up to 6 h for analyses. The results showed that intestinal lymph from rats given L-Gln had increased levels of apolipoprotein B (ApoB) and A-I (ApoA-I), concomitant with an increased spectrum of smaller chylomicron particles. Unexpectedly, L-Gln also increased levels of rat mucosal mast cell protease II (RMCPII), as well as histamine and prostaglandin D In geemokines ,9. Mucosemokines ,11. Depeemokines . Once themokines ,13,14. Iemokines ,16. We cin lymph . And we in lymph ,16. HoweGlutamine (Gln) is an abundant free amino acid in plasma, skeletal muscle, fetal fluids, and milk ,18. It eIn this study, using our well-established conscious lymph fistula rat model, the release of RMCPII (a marker for MMC activation in rats) and other mediators secreted by MMC, as well as intestinal fat absorption in response to a dietary fat load in Gln-treated vs. untreated control rats, were examined. Additionally, the specific effect of L-Gln was further detected by comparing the data to D-Gln supplemented animals.Adult male Sprague-Dawley rats were purchased from Envigo  weighing between 280 and 325 g. Before experimentation, the animals were allowed to acclimate in animal facilities with a 12:12-h light-dark cycle for 2 weeks before the experiments. During this period, they were provided with the standard rodent chow and water ad libitum. The temperature and humidity of the room were maintained at 70° F–74° F and 40–60%, respectively.All surgical procedures were performed as described previously . The aniAfter surgery, the animals were given an intraduodenal infusion of a 5% glucose-saline solution  at a rate of 3 mL/h for 8 h to replenish the loss of fluid and electrolytes due to lymphatic drainage. The infusion was then switched to saline alone, or saline plus 85 mM L-Gln  or 85 mM D-Gln  for overnight infusion until the next morning, respectively. we chose 85 mM Gln because it is within the normal range of solutions of supplemental Gln used in clinical settings ,30. To tLymph triacylglycerol content was measured with a TG assay kit , and phospholipids using Phospholipids C reagents . Both assays were performed according to the manufacturer’s protocols. Protein concentration in lymph was measured by the Bradford method .Lymphatic outputs of apolipoproteins were measured by Western blot, as we described previously ,32. BrieCarbon-coated formvar film on a 400-mesh copper grid  was floated on a drop of the lymph sample. The grid was dried with filter paper and briefly added with a drop of 2% phosphotungstic acid solution (pH 6.0). 1-h lymph samples were not diluted and added to grids as described. Standard beads (200 nm) were used for calibration . The samples were examined with a transmission electron microscope . Images were documented with an AMT advantage Plus CD camera, as described previously ,34. We mDue to termination of the rat mucosal mast cell protease II (RMCPII) enzyme-linked immunosorbent assay (ELISA) kit from Moredun Scientific , lymphatic RMCPII level was measured by Western blot as we described previously in our Gastroenterology paper .2 (PGD2) levels were measured by enzyme-linked immunosorbent assays (ELISA). Lymphatic histamine level was determined with ELISA kits from Neogene . Lymphatic PGD2 level was measured using the PGD2 express EIA kit . All assays were performed according to the manufacturer’s instructions.Lymphatic histamine and prostaglandin Dp < 0.05.Data shown are mean values ± SEMs. To compare the groups throughout the 6-h infusion period, 2-way repeated-measures ANOVA with Tukey posttest analyses were used. A T-test was used for other analyses when comparing only 2 groups. The analyses examined the difference between groups as well as among different time points within the groups. Differences were considered significant at p > 0.05) in lymph flow rates among the three groups of rats, except D-Gln-treated rats had a reduced lymph flow at one time point (3 h) (p < 0.05). These data indicate that the glutamine did not significantly affect the lymph flow rate of the lymph fistula rats. The lymphatic protein outputs increased in all three groups of rats following the bolus lipid meal  during the 6 h of the experiment. As shown in p < 0.05) while not significantly different from the vehicle animals (p > 0.05) at the fifth and sixth hours, respectively.As shown in p < 0.05) and by ~1.9-fold compared to the D-Gln animals (p < 0.01) at the first hour after the feeding of a lipid bolus dose, respectively. ApoB output was stimulated by L-Gln, and this action was specific since D-Gln has no effect. Another fascinating observation was that lymphatic ApoA-I output was also doubled in the first hour following lipid infusion with L-Gln . Again, this effect was specific to L-Gln and not shared by D-Gln. Lastly, p < 0.05) compared with the vehicle controls. Thus, it would reply that L-Gln may be used to stimulate the production of smaller particles by the intestinal epithelial cells during fat absorption. The small particles may metabolize differently from the larger chylomicron particles produced by the animals given lipid only or lipid plus D-Gln.There was no significant difference in the lymphatic TG output between the L-Gln and the vehicle control animals, yet there were more ApoB48 being transported in the lymph of the L-Gln animals than the vehicle controls during the 1st h following the bolus lipid dose. Since there is only one ApoB48 per CM particle, it would imply the L-Gln animals secrete overall smaller CM particles than the vehicle controls. p < 0.01) and at 2 h  and the D-Gln animals at hour one  and at hour two . Similarly, the lymphatic RMCPII secretion was activated significantly more in the L-Gln animals than in the D-Gln animals, and the difference was p < 0.01 for hour 1 and p < 0.05 for hour 2. p < 0.01) in the L-Gln-treated rats (p < 0.01) and the D-Gln animals (p < 0.01). No significant difference was detected between the three groups from hours 4–6th of the experiment (p > 0.05).Our previous study showed that active dietary fat absorption activates the intestinal mucosal mast cells to release RMCPII ,16, a spted rats C during p < 0.05, and vs. 10.98 ± 2.66 ng/mL in D-Gln group, p < 0.01, respectively) after the bolus infusion but returned to the fasting levels by the second hour. It should be noted that the histamine response in lymph bears resemblance but is not identical to the lymphatic RMCPII secretion.An intraduodenal bolus infusion of a 20% Intralipid emulsion alone dramatically increased the release of histamine in lymph after 1 h, as seen in the vehicle controls . The L-G2 [2 was similar to the lymphatic RMCPII and histamine secretion. As shown in 2 during the fat absorption much more than the vehicle controls at hour one  and the D-Gln animals at hour one  and hour two , respectively. However, there was no significant difference between the L-Gln and vehicle groups at hour two . Moreover, the time course of PGD2 concentration in lymph coincided with that of RMCPII release, peaking at 1 h followed by a slow decline reaching a steady-state concentration during hour five. As shown in p < 0.01) in L-Gln-treated rats during the first 3 h than in both the vehicle controls and the D-Gln animals. The total PGD2 outputs within 3 h post-infusion was significantly increased in the L-Gln-treated rats  compared with vehicle controls  and the D-Gln animals . No significant difference (p > 0.05) was detected between the three groups from hours four to six of the experiment.In addition to the preformed mediators RMCPII and histamine, we have also observed in the previous study that fat-activated mast cells also released de novo synthesized mediators such as lipid mediator PGD2 , the maj2 . Thus, w2 in response to dietary fat. All of these effects were specific to L-Gln and not shared by D-Gln. In the present study, we, for the first time, demonstrated a link between the amino acid and the activation of intestinal MMCs in the process of active fat absorption.Using the conscious lymph fistula rat model, we found that the intraduodenal administration of 85 mM L-Gln significantly increased the lymphatic output of ApoB and ApoA-I, which usually both remain constant in response to dietary fat. The finding of ApoB is particularly interesting since there is one ApoB48 molecule associated with each chylomicron particle ,44. As wThough technically difficult, the conscious lymph fistula rat model is by far the most direct and sensitive model to study the in vivo secretion of the inflammatory factors by the intestine immune cells. Firstly, this model allows us to collect lymph directly after the fat stimuli. Since the lymph has not entered the circulation, the secretions contained in lymph are nascent and have not been metabolized by peripheral organs or the liver ; therefoDietary fats are digested and absorbed by the enterocytes and packaged into chylomicrons . ChylomiIn contrast, the lipid outputs and the output of apolipoproteins are quite different. As shown in 2 also doubled in L-Gln-treated rats. These dramatic rises were not caused by a change in flow rate, because the lymph flow rate was comparable among the groups. Interestingly, infusion of an 85 mM solution of the stereoisomer D-Gln did not cause the changes of the RMCPII, histamine, and PGD2 compared with the vehicle controls, suggesting that the effect of L-Gln is specific and probably due to a cellular action of L-Gln. Based on the data, we propose that increased chylomicron production is linked to the L-Gln-promoted activation of MMCs in response to lipid load. Evidence from other studies suggested that intestinal permeability is increased by mast cell mediators, such as histamine, TNF-α, and IL-6 [We have previously demonstrated that dietary fat can activate the intestinal MMCs to release RMCPII . We confand IL-6 ,48,55. Iand IL-6  and can and IL-6 . TherefoOn the other hand, the increased chylomicron formation may be responsible for MMCs activation. During the absorption of fat, the gut basement membrane acts as a temporary barrier preventing the chylomicrons from entering the lamina propria. However, the accumulation of chylomicrons in the intercellular space undoubtedly puts physical stress on the tight junctions, causing breaks in the gut basement membrane, which consequently facilitates the transport of chylomicrons ,58,59. MAlthough some studies showed favorable effects ,61, the 2 (9381.69 ± 971.3 pg/mL vs. 5483.44 ± 705.52 pg/mL in the vehicle controls), which may be related to the intestinal production of apolipoproteins and the formation of smaller chylomicrons. Our findings provide new insight into the role of Gln in gut inflammation and intestinal fat absorption and also remind us of some new sights as well as considerations in our use of L-Gln to promote human intestinal health in normal and disease states.In conclusion, our studies showed that L-Gln specifically promotes intestinal MMCs activation after the ingestion of fat in rats, including RMCPII (2.02~2.29-fold compared with vehicle controls), histamine (26.65 ± 4.95 ng/mL vs.16.07 ± 1.38 ng/mL in the vehicle controls), and PGD


SARS-CoV-2 variant tracking is key to the genomic surveillance of the COVID-19 pandemic. While next-generation sequencing (NGS) is commonly used for variant determination, it is expensive and time-consuming. Variant-specific PCR (vsPCR) is a faster, cheaper method that detects specific mutations that are considered variant-defining. These tests usually rely on specific amplification when a mutation is present or a specific melting temperature peak after amplification.A discrepant result between vsPCR and NGS was found in seventeen SARS-CoV-2 samples from Galicia, Spain. A cluster of BA.1 Omicron SARS-CoV-2 variant showed a BA.2-like melting temperature pattern due to a point mutation (C21772T) downstream the deletion of the spike amino acids 69/70. As the 69/70 deletion is widely used for differentiation between BA.1 and BA.2 by vsPCR, C21772T can cause BA.1 samples to be misinterpreted as BA.2. Over a thousand BA.1 sequences in the EpiCoV database contain this mutation.To our knowledge, this is the first case of a point mutation causing a vsPCR algorithm to misclassify BA.1 samples as BA.2. This is an example of how mutations in the probe target area of vsPCR tests based on melting curve analysis can lead to variant misclassification. NGS confirmation of vsPCR results is relevant for the accuracy of the epidemiological surveillance. In order to overcome the possible impact of novel mutations, diagnostic tools must be constantly updated.The online version contains supplementary material available at 10.1186/s12985-022-01883-2. During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, epidemiological surveillance teams have relied on next-generation sequencing (NGS) to track the spread of new variants and mutations. As a cheaper, faster alternative, commercial providers have developed assays in order to detect mutations that define a specific variant  , 2. ThesHere, we report a cluster of seventeen SARS-CoV-2 strains assigned by next generation sequencing as BA.1 that were erroneously designated by one vsPCR assay as BA.2 due to a detection failure of the 69/70 deletion. Our objective was to look for point mutations in the region surrounding the deletion that could explain this discrepancy. This is a new example of how point mutations can alter variant determination results via vsPCR, and of the potential implications for vsPCR-based SARS-CoV-2 surveillance.In March 2022, 17 patients diagnosed with SARS CoV-2 infection at the Complexo Hospitalario Universitario de Vigo  showed discrepant SARS-CoV-2 variant results between an automated vsPCR assay and NGS. For the vsPCR assay, RNA extraction was performed with GXT 96 × 3 Extraction kit v1.0  on the GenoXtract ® fleXT instrument (Bruker) and PCR was performed with Fluorotype SARS-CoV-2 varID Q  on the FluoroCycler XT (Hain Lifescience) instrument according to manufacturer’s instructions. The mutation A67V (C21762T) upstream the 69/70del is usually present in BA.1 variants. In consequence, tests like the Hain test used for this study updated their reagents to detect the specific combination of A67V + 69/70del , 5. A faFor NGS, RNA extraction from the same samples was performed using QIASymphony DSP Virus Pathogen Midi kit  according to manufacturer’s instructions. Whole-Genome Sequencing was performed using the COVIDSeq Assay Kit . Sixteen samples were sequenced by MiSeq using MiSeq Reagents v3 (Illumina Inc.) and a Hamilton instrument for automated library preparation, while one sample was sequenced on an iSeq 100 using iSeq 100 I1 Reagents (Illumina Inc.) after manual library preparation. Bioinformatic analysis was performed with a custom made pipeline and UShER genome was used for variant determination (Supplementary data 1). Upon NGS analysis, the samples were characterised as Omicron BA.1.1.14 with the synonymous mutation C21772T immediately downstream the 69/70del. We suggest that this mutation prevented the detection of the 69/70del. Deletion 69/70 leads to the loss of the amino acids histidine (H) and valine (V). Given that ATA, ATC and ATT all translate into isoleucine (I), mutation C21772T did not cause a substitution in the amino acid sequence (Table Mutation C21762T (characteristic of BA.1 subvariant) and mutation C21772T (described in this paper) are highlightedhttps://cran.r-project.org/) using the packages ggtree Contact tracing showed that 10 sequences pertained to high school students, and 4 of them were epidemiologically related. All GISAID accession IDs for these 17 sequences are listed in Supplementary Table 1. It is noteworthy that, upon alignment against the Wuhan-Hu-1 reference, some alignments misplace the 69/70del, and thus, the mutation is numbered as A21766T instead of C21772T (Supplementary Table 2). That is the case of databases such as CoVSpectrum and Nextclade [In order to confirm the discrepancy between NGS and vsPCR, the 17 BA.1 + C21772T samples were re-tested in the Hain assay. In this second test, the QIAsymphony nucleic acid extract obtained for NGS was used, in order to avoid any discrepancies caused by the different extraction methods. The second extract gave the same false-negative result and BA.2 interpretation  and BA.2 (69/70del negative) subvariants. Some commercial tests rely on the false-negative result on S-gene to detect BA.1 (SGTF) , whereasCommonly, RT-PCR tests for detection of SARS-CoV-2 are based on fluorescent probes binding to a specific amplicon, although some assays have been developed using dsDNA binding dyes such as SYBR Green , 12. ExaAmongst some of these reports, a pre-Omicron case report showed that a single point mutation in the probe binding sequence can interfere with SARS-CoV-2 detection on a RT-PCR diagnostic test . Other cSingle nucleotide polymorphisms (SNPs) affecting test results have also been shown for melting curve-based assays. For instance, as presented in this publication, mutation C21762T of BA.1, overlapping with the probe sequence in both Hain and VirSNiP , 15, cauRegarding the case studied in this publication, the deletion 69/70 and the surrounding region is targeted by the fluorescent probe and not the primers, according to the instructions manual of both tests , 15 and As of May 16th, there are 1315 sequences from BA.1 with the 69/70del and C21772T mutation, belonging to different BA.1 subvariants . This isOur example is only one of many challenges arising for variant tracking with PCR-based subvariant surveillance for the Omicron wave of SARS-CoV-2: as total cases increase, so do the chances of diagnostic PCR tests affected by a novel mutation such as the ones mentioned above. Also, the recently discovered BA.4 and BA.5 Omicron subvariants show a new mutational pattern not expected by the interpretation software, making variant determination with vsPCR increasingly complex. For example, mutation A67V makes possible the differentiation between BA.1 and BA.4/5, as explained in this report. However, BA.4 and BA.5 are genetically identical in the area surrounding 69/70del, so other targets are needed for lineage determination. Finally, recombinant SARS-CoV-2 variants such as PANGO lineage XD or XE, which combine AY.4/BA.1 and BA.1/BA.2 respectively, increase the complexity for taxonomic classification of the virus using methods based in the detection of combinations of single point mutations.As Omicron subvariants show a high number of spike mutations, and given all the complexities mentioned, we recommend commercial kit providers to increase their available targets in order to obtain a more specific differentiation between SARS-CoV-2 lineages and sublineages with vsPCR assays. Increasing the number of targets in a single reaction is challenging, given that too many fluorophores would lead to overlapping emission spectra and suboptimal results. While this has been solved by making two reactions per sample by some manufacturers, it can be more costly and time-consuming. Another commercial approach is to offer a wide range of targets and fluorophores in order to be prepared for the detection of any new variant that arises. A constant update of the available targets and periodic checks of their performance with circulating SARS-CoV-2 isolates are key to maintaining optimal epidemiological surveillance.The speed in the detection of new mutations and variants is crucial in order to prepare an adequate response to the new circumstances of the COVID-19 pandemic. For instance, the appearance of variants Delta and Omicron caused a change in strategies as these variants were responsible for an increased number of cases and hospitalisations due to increased transmissibility and immune escape –23. In tBelow is the link to the electronic supplementary material.Supplementary Material 1